Eukaryotic fatty acid desaturation is catalyzed by O2-dependent multiprotein complexes. The most abundant unsaturated fatty acid, oleic acid, is produced by either the soluble plant stearoyl-acyl carrier protein delta-9 desaturase (delta9D) or the membrane bound mammalian stearoyl-CoA delta9 desaturase. Unsaturated fatty acids are essential membrane components and serve as precursors to leukotrienes, plasmologens, prostaglandins and thromboxanes. They are also found in many commercial products. However, despite the importance of unsaturated fatty acids, the reaction mechanism of this critical biosynthetic process is essentially unknown. The long term goal of our research is to define this reaction mechanism at the molecular level using the first abundantly available recombinant desaturase, the delta9D from castor plant. The PI has recently shown that the delta9D is a member of the newly identified class of enzymes containing mu-oxo-(R//H)-bridged diiron clusters. Ribonucleotide reductase and methane monooxygenase also use iron-oxo clusters to catalyze two distinct types of chemical reactions requiring O2-activation. Our Preliminary Studies show that the delta9D catalyzes oxidative desaturation by using the iron-oxo cluster in a third, and presently unknown, mechanism of O2-activation. We will use biochemical, kinetic and spectroscopic methods to clarify the role of the iron-oxo cluster in the biosynthesis of unsaturated fatty acids. The Specific Aims of our research will be to determine the following: l) What physical properties distinguish the iron-oxo cluster of the delta9D from those of other iron-oxo enzymes; 2) What type of reactive intermediate is formed when the delta9D cluster reacts with O2; and 3) How is the delta9D reactive intermediate used for desaturation catalysis. These studies will define the role of the iron-oxo cluster in fatty acid biosynthesis and provide new insight into the mechanism of oxidative desaturation by eukaryotes. Both of these outcomes will help to further define the principles which govern O2-activation by the proteins containing iron-oxo clusters.